In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for tissue-dependent alternative splicing of two nonmuscle MHC II (NMHC II) genes, NMHC II-B and NMHC II-C. Our past study revealed that a new RNA binding protein family, the Rbfox family, plays a critical role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. In this report, we focus on function of Rbfox proteins. Rbfox proteins contain a single conserved RNA recognition motif (RRM) in the central region of the molecule and are known to bind specifically to an RNA penta(hexa)nucleotide (U)GCAUG. There are three genes for Rbfox family proteins in mammals, Rbfox1, Rbfox2 and Rbfox3. Rbfox1 is expressed in brain and striated muscles whereas Rbfox2 is expressed in various tissues including brain and muscles. Notably, Rbfox3 expression is restricted to neural tissues. Exogenous expression of each of Rbfox1, Rbfox2 and Rbfox3 in cultured cells is capable of activating the neuron-specific exon through its binding to the UGCAUG element located in the downstream intron. However biochemical analyses of mouse brain cells sorted by Rbfox antibody staining and histological analyses demonstrated that the expression level of the neuron-specific splice variant of NMHC II-B mRNA correlated better with the level of Rbfox-3 expression rather than with that of Rbfox-1 or Rbfox2 expression. These observations suggest that Rbfox3 contributes neuron-specific splicing of NMHC II-B mRNA, although brain expresses all three Rbfox proteins. Next we extended our research to study the biological function of Rbfox3. We made use of mouse embryonic carcinoma P19 cells which are capable of differentiating into neuronal cells following retinoic acid treatment. Neuronal differentiation of P19 cells can be monitored by outgrowth of a long axon-like extension which contains an axonal marker, phosphorylated neurofilaments. During neuronal differentiation, expression of Rbfox3 is induced whereas undifferentiated P19 cells do not express Rbfox3. Rbfox1 is barely detected under both undifferentiated and differentiated conditions and the Rbfox2 expression level is unchanged before and after differentiation. The shRNA-mediated knock-down of Rbfox3 results in a decrease in axon-like extensions and an almost complete elimination of phosphorylated neurofilaments. These results indicate that Rbfox3 is required for neuronal differentiation of P19 cells. To understand molecular mechanism for Rbfox3 function in neuronal differentiation of P19 cells, we carried out Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) followed by high-throughput RNA sequencing. This analysis identified transcriptome-wide target RNAs of Rbfox3 in neuronally differentiated P19 cells. Unexpectedly, we found that Rbfox3 interacts with a much broader range of RNA sequences than the previously determined UGCAUG motif, and that miRNA hairpin loci are over-represented in Rbfox3 targets. Induced expression and depletion of Rbfox3 led to changes in the expression levels of a subset of PAR-CLIP-detected miRNAs in P19 cells. Rbfox3 changes expression levels of mature miRNAs and pre-miRNAs but not pri-miRNAs, suggesting that Rbfox3 affects processing of pri-miRNAs to pre-miRNAs. In vitro biochemical analyses revealed that Rbfox3 binds directly to pri-miRNAs, which lack the UGCAUG motif, and regulates the recruitment of the microprocessor complex to pri-miRNAs. Depending on the pri-miRNA contexts, Rbfox3 binds either the terminal loop or the stem region of the pri-miRNAs and either promotes or inhibits respectively, the cleavage of the pre-miRNAs. Our study proposes a novel function for Rbfox3 in miRNA biogenesis.